Abstract：
Under the background of food security, using non-grain feed instead of corn-soybean-based feed is an effective measure to alleviate the food-feed competition. While, non-grain feeds are often rich in fiber, which cannot be digested by non-ruminants. Producing heterologous enzymes in non-ruminants to improve cellulose utilization rate is a new research strategy by transgenic technology. In this study, porcine transthyretin (TTR) promoter, signal peptide-coding sequence (CDS), Saccharomycopsis fibuligera β-glucosidase gene (BGL1)-CDS, 6×His sequences fragments were fused into pGL3-control vector to generate transgenic vector. Then, transgenic mice were generated by pronuclear microinjection of the linearized expression vectors. Transgenic mice and their offspring were examined by PCR-based genotyping and copy number variation. Results showed that BGL1 was successfully integrated into the mouse genome and transmitted stably. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and β-glucosidase activity assay demonstrated that BGL1 was specifically expressed in the liver, and β-glucosidase activity significantly increased. In addition, liver weight index, cellular morphology, and collagen fiber content of the liver showed that exogenous gene insertion did not cause any lesions to live. Taken together, our findings suggest that β-glucosidase driven by TTR promoter was specifically expressed in the liver of transgenic mice.
摘要：
在粮食安全的大背景下,使用非谷物饲料代替玉米大豆饲料是缓解粮食与饲料竞争的有效措施。同时,非谷物饲料通常富含纤维，不能被非反刍动物消化。在非反刍动物中产生异源酶以提高纤维素利用率是转基因技术的新研究策略。在这项研究中,猪甲状腺素运载蛋白(TTR)启动子,信号肽编码序列（CDS），纤维酵母菌β-葡萄糖苷酶基因(BGL1)-CDS,将6xHis序列片段融合到pGL3对照载体中以产生转基因载体。然后，通过线性化表达载体的原核显微注射产生转基因小鼠。通过基于PCR的基因分型和拷贝数变异检查转基因小鼠及其后代。结果表明，BGL1成功整合到小鼠基因组中并稳定传播。此外，逆转录-聚合酶链反应(RT-PCR),西方印迹,和β-葡萄糖苷酶活性测定表明，BGL1在肝脏中特异性表达，β-葡萄糖苷酶活性显著增加。此外,肝脏重量指数,细胞形态学,肝脏的胶原纤维含量表明，外源基因的插入没有引起任何病变的存活。一起来看,我们的发现表明，由TTR启动子驱动的β-葡萄糖苷酶在转基因小鼠的肝脏中特异性表达。
